The present invention, in some embodiments thereof, relates to crystal structures and structure-based drug design and, more particularly, but not exclusively, to methods of designing species-specific antimicrobial agents based on crystal structures of pathogenic ribosomal subunits.
The clinical usage of the currently available antibiotics is becoming ever more limited due to the capability of pathogens to undergo mutations, the phenotype thereof minimizes or abolishes binding interaction of the antibiotics to the molecular target in the pathogen. The emergence of bacterial resistance to antibiotics threatens regression to the pre-antibiotic era as the treatments of infections with the available arsenal of clinically used antibiotics have suffered from the appearance of multidrug-resistant strains. Consequently, many hospital-acquired infections are currently caused by highly resistant bacteria such as methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant Staphylococcus aureus (VRSA), Gram positive versatile and aggressive pathogens that are among the most worrisome pathogenic bacteria.
Among the existing antibiotics, many target the fundamental process of protein biosynthesis, mostly by interrupting bacteria's ribosomal activity. A large number of antibiotics target the universal cellular multi-components RNP (RNA-protein) particles translating the genetic code into proteins.
The increasing epidemiology of Staphylococcus aureus (SA) infections revealed that most of SA strains have 5-6 copies of the operon of the main rRNA (ribosomal RNA) component, namely the 23S rRNA. The fact that SA has 5-6 operon copies is most likely correlated with the finding that resistance that is caused by a single RNA mutation is accumulative.
It has been observed that in SA, the commonly occurring resistance to ribosomal-active antibiotics is acquired by a single-nucleotide mutation in the 23S large ribosomal subunit rRNA. SA resistant mutations are also associated with the region of rProtein (ribosomal protein) L3 that is located in proximity to the peptidyl transferase center (PTC) and rProteins L4 and L22 that reach the exit tunnel.
Infections caused by SA are typically treated by several antibiotics, including the ribosomal-active antibiotics linezolid, telithromycin and pleuromutilins such as BC-3205, the structures of which are presented hereinbelow, which bind to the large ribosomal subunit.
Linezolid is a synthetic drug which has been approved by FDA on April 2000 for treating Gram positive pathogen infections. It belongs to the class of oxazolidinones and was designed to bind at the PTC. Since linezolid is a synthetic drug, no pre-existing resistance mechanisms were known prior to its use, although resistance to other ribosomal-active antibiotics that target the same site has been identified. However, it was expected that emergence of resistance to this drug would occur rather slowly. Despite these expectations, S. aureus linezolid resistance, acquired by a specific 23S point mutation, referred to as G2576U (E. coli numbering is used throughout), was reported a year after its approval for treatment. Since linezolid is often used as the last line of defense against multidrug-resistant bacterial infections, a resistance to linezolid in clinical isolates was reported to be rather rare, and on 2010, it was reported to occur in less than 1% of SA isolates.
The observed G2576U resistance mutation is in accord with the crystal structures and with a model of ribosome-linezolid complexes [Wilson, D. N. et al., P.N.A.S. USA, 2008, 105(36), p. 13339-44; Ippolito, J. A. et al., J Med Chem, 2008, 51(12), p. 3353-6; and Leach, K. L. et al., Molecular Cell, 2007, 26(3), p. 393-402], which indicate that the drug binding site is composed of 23S rRNA nucleotides that form the inner shell of the PTC, thus perturbing the correct positioning of tRNAs on the ribosome. Interestingly, the G2576U resistance mutation was found to be associated with linezolid resistance in the multi-drug resistant bacteria Enterococcus faecium and Enterococcus faecalis and in S. pneumonia, as well as the tuberculosis diagnostic tool Mycobacterium smegmatis. 
Telithromycin is a ketolide antibacterial agent/drug (antibiotic) that is structurally related to macrolides, and which has been developed specifically to provide optimal therapy for the treatment of respiratory tract infections (RTI) caused by either typical or atypical respiratory pathogens. Ketolides possess two innovative structural modifications, a 3-keto group and a large N-substituted C11, C12-carbamate side chain. Telithromycin has an additional long alkyl-aryl arm. Telithromycin have showed potent in vitro activity against S. pneumoniae, including strains resistant to macrolide-lincosamide-streptogramin B ketolide (MLSBK) and sub-inhibitory concentrations of telithromycin have been found to inhibit MRSA in vitro.
Crystal structures showed that telithromycin binds to the large ribosomal subunit at the macrolide binding pocket in the ribosome's exit tunnel [Berisio, R. et al., J Bacteriol., 2003, 185(14), p. 4276-9; Tu, D et al., Cell, 2005, 121(2), p. 257-70; Dunkle, J. A. et al., Proc Natl Acad Sci USA, 2010, 107(40), p. 17152-17157; and Bulkley, D. et al., Proc Natl Acad Sci USA, 2010, 107(40), p. 17158-63], and that its flexible alkyl-aryl arm is pointing in different directions in different species.
Pleuromutilin and its derivatives are antibacterial agents (antibiotics) that inhibit protein synthesis in bacteria by binding to the peptidyl transferase component (PTC) of the 50S subunit of ribosomes. Pleuromutilin is a natural product of the fungi Pleurotus mutilus (also known as Clitopilus scyphoides), which has been used as a base for the synthesis of several semi-synthetic antibacterial agents, designed for clinical utilization by targeting eubacterial ribosomes. Members of this class of antibiotics, collectively referred to herein and in the art as Pleuromutilins, which includes retapamulin, valnemulin and tiamulin and some investigational drugs such as azamulin and BC-3781, all exhibit a tricyclic mutilin core, a C21 keto group, essential for antimicrobial activity, and various substituents at the C14, most of which are extensions of diverse chemical nature. Some of these semi-synthetic antibacterial agents are already in clinical use and exhibit elevated activity over a broad spectrum of pathogens.
Retapamulin belongs to the group of C14-sulfanyl-acetate derivatives of Pleuromutilin, and was approved for use as a topical antibiotic on 2007. Retapamulin was shown to possess potent activity against Gram positive pathogens, and a low propensity to develop resistance. Thus, all strains of Staphylococcus aureus and Streptococcus pyogenes were susceptible to retapamulin at a minimal inhibition concentration (MIC) of 0.5 gram/ml. Other C14-sulfanyl-acetate derivatives of Pleuromutilin, valnemulin and tiamulin, were approved for veterinary clinical use.
Recent advances in pleuromutilin's chemistry yielded several new compounds as potential antibacterial agents. Among them are BC-3205, which is a semi-synthetic pleuromutilin derivative that was developed for oral treatment of skin and skin structure infections (SSSI) and community-acquired pneumonia (CAP), as well as BC-3781 and BC-7013, all of which by Nabriva Therapeutics AG, Vienna, Austria. BC-3205 acts against SA with a MIC of 0.06 μg/ml, is 16-32-fold more potent than linezolid against SA and is therefore considered as highly potent antibacterial agent.
The available crystal structures of complexes of the large ribosomal subunit from a non-pathogenic model bacterium, D50S, with various pleuromutilin compounds, namely tiamulin, retapamulin, SB-264128 and SB-571519, revealed that these compounds are bound to the large ribosomal subunit at the PTC. In all cases the cores of these compounds are placed in a similar fashion at the A-site, and the C14 extensions of these compounds are pointing towards the P-site, thus, directly inhibiting peptide bond formation. As the PTC is almost fully conserved, the pleuromutilin's efficient inhibitory modes are attained by exploiting the ribosomal intrinsic functional flexibility for induced-fit and remote conformational rearrangements that result in tightening up the binding pockets [Schlünzen, F. et al., Molecular microbiology, 2004, 54, p. 1287-1294; and Davidovich, C. et al., Proceedings of the National Academy of Sciences, 104, p. 4291-4296].
Species specificity in relevance to emergence of resistance to antibiotics in bacteria has been observed; however, so far all available structural information on ribosomal-antibiotics interactions has been obtained from ribosomal particles and subunits of non-pathogenic bacteria, which only emphasized the common traits. Previous studies which compared structures of ribosomes from different kingdoms of life [Petrov, A. S. et al., P.N.A.S. USA, 2014, 111(28), p. 10251-10256] have prompted suggestions concerning pathways in ribosome evolution.
Previous structural studies on antibiotics' modes of binding and bioactivity were based only on the available ribosomal crystal structures, which were of eubacteria suitable to mimic pathogens under clinical relevant conditions. These include D50S of Deinococcus radiodurans [Schluenzen, F. et al., Nature, 2001, 413, p. 814-21], T70S of Thermus thermophilus [Voorhees, R. M. et al., Nat Struct Mol Biol., 2009, 16(5), p. 528-33] and E70S from the non-pathogenic strain of Escherichia coli [Schuwirth, B. S. et al., Science, 2005, 310(5749), p. 827-834]. Results of these studies provided useful insights for common traits of the mode of action of antibiotics, such as binding at ribosomal functional sites, e.g. the PTC or the protein exit tunnel; illuminated structural bases for the distinction between patients (eukaryotes) and pathogens (eubacteria) despite the high conservation of the ribosomal functional sites; and shed light on antibiotics synergism and the general principles of resistance and cross resistance.
Based on the similarity in their sequences, the structures of ribosomes from pathogens are expected to resemble ribosomes from other eubacteria; however, species specificity in clinically relevant properties, particularly in the modes of acquiring antibiotic resistance, has been identified [Wilson, D. N., Ann. N.Y. Acad. Sci., 2011, 1241, p. 1-16], and it has been shown that small structural differences between bacterial species could affect the drug binding [Yonath, A., Mol Cells, 2005, 20, p. 1-16].
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